STING/stimulator of interferon genes

ADU-S100's free base shape is erratic. ADU-S100 ammonium salt increases stability and lipophilicity in comparison to endogenous and pathogen-derived cyclic dinucleotides (CDNs), hence stimulating a noteworthy upregulation of STING signaling [1]. In human monocytes THP-1, ADU-S100 produced more type I IFN than CDA. On the other hand, all five hSTING alleles, including the refractory hSTINGREF and hSTINGQ, are efficiently activated by mixed disulfide-linked CDN derivatives (ML RR-CDA, ML RR-S2 CDG, and ML RR-S2 cGAMP). bit gene. When compared to endogenous ML cGAMP and the TLR3 agonist Poly I:C, ADU-S100 elicited the highest production of IFN-β and the proinflammatory cytokines TNF-α, IL-6, and MCP-1 on a molar equivalent basis. It was also shown that ADU-S100 stimulated TBK1 and IRF3 phosphorylation in mouse bone marrow macrophages (BMM) as well as STING aggregation. In comparison to ML cGAMP, ADU-S100 generates noticeably larger amounts of IFN-α [1].

ADU-S100 outperformed endogenous ML cGAMP in its anti-tumor control capacity. In B16 tumor-bearing mice, a dose response study of ADU-S100 compounds was carried out to ascertain the best anti-tumor dose level that would maximize tumor antigen-specific CD8+ T cell responses and increase long-term survival to 50%[1].

Luciferase Assay[1]
104 HEK293T cells were seeded in 96-well plates and transiently transfected with human IFN-β firefly reporter plasmid(Fitzgerald et al., 2003) and TK-Renilla luciferase reporter for normalization. The following day, cells were stimulated with 10 μM of eachADU-S100 or 100 μg/ml DMXAA using digitonin permeabilization (50 mM HEPES, 100 mM KCL, 3 mM MgCl2, 0.1 mM DTT, 85 mM Sucrose, 0.2% BSA, 1 mM ATP, 0.1 mM GTP, 10 ug/ml digitonin) to ensure uniform uptake. After 20 min, stimulation mixtures were removed and normal media was added. After a total of 6 hours, cell lysates were prepared and reporter gene activity measured using the Dual Luciferase Assay System on a Spectramax M3 luminometer.

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Differential Scanning Fluorimetry[1]
Thermal shift assays were performed as (Cavlar et al., 2013). Assays were conducted with STING ligand binding domain at 1 mg/ml with or without various ADU-S100 at 1 mM in 20mM Tris-HCL, 150 mM NaCl, pH 7.5 and 1:500 dilution of SYPRO Orange Dye. The fluorescence as a function of temperature was recorded in a CFX 96 real time PCR machine reading on the HEX channel EX 450–490 EM 560–580 nm. The temperature gradient was from 15–80°C ramping 0.5°C per 15 seconds. Curves were fit to a Boltzmann sigmoidal to establish the midpoint of thermal unfolding (Tm).

BM-DCs from WT or STING−/− mice were stimulated with 25 μg/ml DMXAA or 100 ng/ml LPS for 4 hours. Total RNA was isolated using the RNeasy® kit and incubated with Deoxyribonuclease I, Amplification Grade. cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit and expression of cytokines was measured by real-time qRT-PCR using specific primers/probes for mouse INF-β, TNF-α, IL-6 and IL12p40, and pan-specific primers were to quantify expression of the IFN-α family. Primer sequences are listed in Table 1 in Supplementary Materials. PCR reactions were performed in the 7300 Real Time PCR system. The results are expressed as 2−ΔCt using 18s as endogenous control.

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WT BMM were stimulated with ADU-S100 at 5 μM in HBSS with the addition of Effectene transfection reagent (per kit protocol). Human PBMCs were stimulated as indicated. Stimulated cells were and assessed by real-time qRT-PCR for gene expression of IFN-β1, MCP-1, TNF-α and IL-6 using the PrimePCR RNA purification and cDNA analysis system, and run on the CFX96 gene cycler. Relative normalized expression was determined by comparing induced target gene expression to unstimulated controls, using the reference genes Gapdh and Ywhaz (mouse) and GusB and Pgk1 (human), genes confirmed to have a coefficient variable (CV) below 0.5 and M value below 1, and thus did not vary with different treatment conditions.

10~6 of B16-SIY tumor cells, 5 × 10~4 B16.F10 tumor cells, 10~5 4T-1 and CT26, or 106 other tumor cells were injected s.c. in 100 μl DPBS or HBSS on the right flank of mice. Following tumor implantation, mice were randomized into treatment groups. When tumors were 100–200 mm3 in volume (5–7 mm wide), either one single or three doses of DMXAA resuspended in 7.5% of NaHCO3, or CDNs formulated in HBSS or vehicle control, were injected IT. Measurements of tumors were performed twice per week using calipers, and the tumor volume was calculated with the formula: V= (length × width2)/2. In some experiments, tumor-free survivors were rechallenged with tumor cells on the opposite flank several weeks after the injection of the primary tumor. Naïve mice were used as controls. For the contralateral experiments, mice were implanted on both flanks and only one tumor was treated. For the B16 melanoma lung metastasis experiments, mice were implanted on the flank with 5 × 104 cells B16.F10 on day 0, and then injected intravenously with 1 × 105 cells on day 7. Lungs were harvested on day 28. Administration of compounds, measurements of tumors and counting of lung tumors were performed in a blinded fashion.[1]

[1]. Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep. 2015 May 19;11(7):1018-30.

ADU-S100 (MIW815) is a synthetic cyclic dinucleotide (CDN) agonist (activator) of Stimulator of Interferon Genes (STING), a receptor crucial to activate the innate (endogenous) immune system. ADU-S100 (MIW815) activates all known human and mouse STINGs, and effectively induces the expression of cytokines and chemokines, leading to a robust and durable antigen-specific T-cell mediated immune response against cancer cells. DrugBank STING-activating Cyclic Dinucleotide Agonist MIW815 is a synthetic, cyclic dinucleotide (CDN) and agonist of stimulator of interferon genes protein (STING; transmembrane protein 173; TMEM173), with potential immunomodulating and antineoplastic activities. Upon intratumoral administration, the STING agonist MIW815 binds to STING and stimulates STING-mediated pathways. This activates the immune response through the activation of certain immune cells, including dendritic cells (DCs), which induces the expression of cytokines and chemokines, and leads to an antigen-specific T-cell mediated immune response against cancer cells. STING, a transmembrane protein that activates immune cells in the tumor microenvironment, plays a key role in the activation of the innate immune system.

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Spontaneous tumor-initiated T cell priming is dependent on IFN-β production by tumor-resident dendritic cells. On the basis of recent observations indicating that IFN-β expression was dependent upon activation of the host STING pathway, we hypothesized that direct engagement of STING through intratumoral (IT) administration of specific agonists would result in effective anti-tumor therapy. After proof-of-principle studies using the mouse STING agonist DMXAA showed a potent therapeutic effect, we generated synthetic cyclic dinucleotide (CDN) derivatives that activated all human STING alleles as well as murine STING. IT injection of STING agonists induced profound regression of established tumors in mice and generated substantial systemic immune responses capable of rejecting distant metastases and providing long-lived immunologic memory. Synthetic CDNs have high translational potential as a cancer therapeutic.[1]

C20H24N10O10P2S2

690.54

690.06

C, 34.79; H, 3.50; N, 20.28; O, 23.17; P, 8.97; S, 9.29

1638241-89-0

ADU-S100 disodium salt;1638750-95-4;ADU-S100 ammonium salt;1638750-96-5;ADU-S100 enantiomer ammonium salt

White to off-white solid powder

OC1([H])[C@](O[P@](S)(OC[C@](O[C@@H](N2C3=NC=NC(N)=C3N=C2)[C@@H]4O)([H])[C@@]4([H])O5)=O)([H])[C@H](N6C7=NC=NC(N)=C7N=C6)O[C@]1([H])CO[P@]5(S)=O

IZJJFUQKKZFVLH-YBVMPXGUSA-N

InChI=1S/C20H24N10O10P2S2/c21-15-9-17(25-3-23-15)29(5-27-9)19-12(32)13-8(38-19)2-36-42(34,44)40-14-11(31)7(1-35-41(33,43)39-13)37-20(14)30-6-28-10-16(22)24-4-26-18(10)30/h3-8,11-14,19-20,31-32H,1-2H2,(H,33,43)(H,34,44)(H2,21,23,25)(H2,22,24,26)/t7-,8-,11-,12-,13-,14-,19-,20-,41-,42-/m1/s1

(2R,5R,7R,8R,10R,12aR,14R,15R,15aS,16R)-7,14-bis(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dimercaptooctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11]pentaoxa[2,10]diphosphacyclotetradecine 2,10-dioxide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)